ABSTRACT
Duodenal bicarbonate secretion is critical to epithelial protection, nutrient digestion/absorption and is impaired in cystic fibrosis (CF). We examined if linaclotide, typically used to treat constipation, may also stimulate duodenal bicarbonate secretion. Bicarbonate secretion was measured in vivo and in vitro using mouse and human duodenum (biopsies and enteroids). Ion transporter localization was identified with confocal microscopy and de novo analysis of human duodenal single cell RNA sequencing (sc-RNAseq) datasets was performed. Linaclotide increased bicarbonate secretion in mouse and human duodenum in the absence of CFTR expression (Cftr knockout mice) or function (CFTRinh-172). NHE3 inhibition contributed to a portion of this response. Linaclotide-stimulated bicarbonate secretion was eliminated by down-regulated in adenoma (DRA, SLC26A3) inhibition during loss of CFTR activity. Sc-RNAseq identified that 70% of villus cells expressed SLC26A3, but not CFTR, mRNA. Loss of CFTR activity and linaclotide increased apical brush border expression of DRA in non-CF and CF differentiated enteroids. These data provide further insights into the action of linaclotide and how DRA may compensate for loss of CFTR in regulating luminal pH. Linaclotide may be a useful therapy for CF individuals with impaired bicarbonate secretion.
Competing Interest Statement
ZMS received financial compensation from AbbVie for professional consultation unrelated to linaclotide. AbbVie did not pay for, contribute, have any special access to data, or have any involvement in any aspect of the work. Also, received consulting fees from 4D Molecular Therapeutics, which had no involvement in the work or access to any data.
Footnotes
Conflicts of Interest: JBS, AMT, SMA, VVU, JEC, YJ, JG, MOA, OC, CJK: The authors have declared no conflict of interest. ZMS: Received financial compensation from AbbVie for professional consultation unrelated to linaclotide. AbbVie did not pay for, contribute, have any special access to data, or have any involvement in any aspect of the work. Also, received consulting fees from 4D Molecular Therapeutics, which had no involvement in the work or access to any data.
Included additional data supporting the ability of CFTRinh-172 to inhibit forskolin-stimulated CFTR activity in mouse tissue and human duodenal enteroids. Given the broad use of CFTRinh-172 throughout the experiments we feel that this additional data provides greater confidence in interpreting our results. We have performed additional mouse in vitro experiments examining DRAs role in linaclotide-stimulated bicarbonate secretion when CFTR is functional. We have done this by increasing the n of our prior experiments but also by performing new experiments using a different DRA inhibitor. These results show that when CFTR is functional DRA is not necessary for linaclotide-stimulated duodenal bicarbonate secretion CFTR is sufficient. We have updated our conclusions (and manuscript title) accordingly. To better understand how CFTR inhibition (or lack of CFTR expression in cystic fibrosis enteroids) leads to increased brush border expression of DRA we performed new experiments measuring intracellular pH in duodenal enteroids before and after CFTR inhibition. In these experiments we found that CFTR inhibition transiently increased intracellular pH likely due to bicarbonate trapping. This alkalinization is transient with subsequent intracellular pH recovery. Pre-treatment of enteroids with DRA inhibitor altered the intracellular pH recovery.